HPV16 E7 Upregulayed The Expression Of ACP5 By Mediating FoxM1 Through Activating CDK6 In Lung Cancer Cells

Objective: Lung cancer is one of the most common malignancies in the world,accounting for 14% and 12% of all cancer incidence,and 27% and 25% of cancer mortality in males and femals,respectively.Lung cancer,as a well-recognized heterogeneous disease,is influenced by a range of environmental and genetic factors,searching for new oncogene and anti-oncogene is helpful in making the most appropriate and timely diagnosis,and developing effective target treatment in lung cancer patients.Human papilloma viruses(HPVs)are a group of double-stranded DNA viruses known to be the primary cause of cervical cancer.In addition,Syrj?nen,et al.first report that HPV infection was involved in the development of lung cancer.With the progress of cell biology,many studies have demonstrated that high-risk HPV16 was the most common type in lung cancer patients.Currently,the two major viral oncoproteins,E6 and E7,are considered as oncogene in development and progression in lung cancer.In our study,firstly,we examined the ACP expression in ascites,pleural effusion,pericardial effusion and serous effusion,and differentiated the malignance from all samples.Secondly,we examined the expression of HPV16 E7,P16,CDK4/6,Fox M1 and ACP5 in lung cancer lines,investigated the correlation among HPV16 E7,P16,CDK4/6,Fox M1 and ACP5.Therefore,we speculated that Fox M1 upregulayed ACP5 is mediated by HPV16E7-induced CDK6 activation mechanism.Since the HPV16 E7 oncoproteins are dominant during lung cancer development,targeting both the E7 oncoproteins and its associated protein would be a promising approach for the development of therapeutic targets that could provide comprehensive therapeutic effects in HPV-mediated lung cancer.Methods:1.A total of 212 serous effusions(128 pleural effusions,69 ascites,and 15 pericardial effusion)which were collected from No.1 Affiliated Hospital of China Medical University from April.2016 to January.2017,were included in our study.In all samples,128 cases were malignant effusions and 88 cases were benign effusions?2.The H460 cell line was obtained from Shanghai Cell Bank;and the LK2 cell line was a gift from Dr.Hiroshi Kijima.3.ACP staining: All samples were examined by cytology examinationa and ACP staining.ACP staining adopted ACP staining kit.The sensitivity,specificity,and accurace were evaluated in differentiating malignant and benign effusion samples.4.The regulation of HPV16 E7,P16,CDK4/6,Fox M1 and ACP5 in lung cancer lines was investigated using westernblot and real-time PCR.The m RNA levels and expression of HPV16 E7,P16,CDK4/6,Fox M1 and ACP5 were examined by interfering HPV16 E7,P16,CDK4,CDK6 and Fox M1,respectively.Results:1.The staining intensity of ACP in benign effusion was significantly higher than malignant effusion(p<0.01).The positive ACP signals were not found in lymphocytes or granulocytes.2.The sensitivity of ACP staining is significantly higher than cytology examination in differentiating malignant effusion and benign effusion,and the specificity reached to100%.3.The protein expressions of downstream target genes were upregulated by transfecting HPV16 E7 in H460 cell line except CDK4,and the m RNA expression differences of CDK6,Fox M1 and ACP5 were significant.4.The protein expressions of downstream target genes were downregulated by interfering HPV16 E7 in LK2 cell line except CDK4,and the m RNA expression differences of CDK6,Fox M1 and ACP5 were significant.5.The protein expressions and m RNA levels in CDK4,CDK6,Fox M1 and ACP5 were upregulated by interfering P16 in LK2 cell line.6.After interfering CDK4/6 and Fox M1,respectively,their target genes were downregulated,and m RNA levels were no different,indicating that CDK4/6 and Fox M1 positively regulated downstream target genes.Conclusion:1.The cocktail ACP staining is an excellent marker with high sensitivity and specificity to distinguish the carcinoma from the reactive mesothelial cells in the body fluid effusions.The cocktail ACP staining can be used as an impactful tool to complement cytological diagnosis and provide a better guidance for the clinical decision making.2.Taken together,our findings demonstrated HPV16 E7/P16/CDK6/Fox M1/ACP5 pathway was activated in lung cancer progression,and provided the evidence of HPV16 E7 underlying mechanisms in lung cancer.